scut-china-b
Is it possible to turn cancer cells directly into immune cells and make them turn on suicide mode? was the initial source of inspiration for our team. To explore this hypothesis, we first focused on how to design and synthesize a plasmid that could achieve this function.
Melanoma, usually referred to as malignant melanoma, is a highly malignant tumor of melanocyte origin, referred to as malignant black, which occurs most often in the skin, but can also be seen in mucous membranes and internal organs. In recent years, the incidence and mortality rate of malignant melanoma have been increasing year by year, and the age of death is lower compared with other solid tumors. Malignant melanoma lacks specific treatment except for early surgical resection and has a poor prognosis. Therefore, early diagnosis and treatment of malignant melanoma is extremely important.
The tyrosinase promoter is an important regulatory sequence that controls the expression of the tyrosinase gene. The tyrosinase promoter regulates the transcription of the tyrosinase gene by interacting with specific transcription factors. MITF (Melanocyte Inducing Factor): As a key transcription factor, MITF binds directly to the tyrosinase promoter and activates its transcription. Since the content of MITF is much higher in melanoma than in normal cells, with the help of tyrosinase promoter we can achieve specific expression of gene nanomedicine in tumor cells.
Induction of T cell activation and proliferation requires two signals: the first activation signal delivered by the TCR-CD3 complex and the second signal generated by a synergistic co-stimulatory molecule dominated by CD86. In vitro co-stimulation of T cells with antibodies against CD3 and CD86 to mimic the dual signaling of T cell activation has become the most widely used method for T cell activation and expansion. These two important activation signals are effective tools for our T cell activation.
Recruitment of T cells along with activation of T cells is also important in promoting immune response. Immunosuppression of regulatory T cells (Tregs) can be attenuated by increasing the infiltration of CD8+ T cells.IL-2 can activate and expand effector T cells and NK cells, which play a crucial role in anti-tumor immune responses. IL-2 also supports regulatory T cells (Treg cells), helping to maintain immune tolerance and prevent autoimmunity. In MHC class I-deficient cancers, the use of IL-2 can restore immune cell infiltration in the tumor microenvironment, improving the immunological context and enhancing therapeutic effects. IL-2 can be used in combination with other therapeutic approaches such as tumor-targeting monoclonal antibodies, chemotherapy, and radiotherapy to improve treatment outcomes. Compared to traditional high-dose IL-2 therapy, the use of engineered IL-2 products (such as low-dose IL-2 or specifically targeted IL-2) can reduce non-specific toxicities and improve the safety profile of the treatment.
We designed tyrosinase promoter-driven NPTyr-αCD3-CD86- mut IL-2 to specifically express anti-CD3 scFv and anti-CD86 on the surface of tumor cells while mobilizing tumor cells to secrete mut IL-2, a cytokine for T-cell proliferation and survival, to activate T-cells and enhance immunotherapy.
We extracted plasmids from E. coli and designed primers and plasmids using snapgene. Finally, we constructed various plasmids such as pU57-Tyr-mut IL-2, pU57-Tyr-αCD3-CD86, pU57-Tyr-αCD3-CD86-mut IL-2, pU57-Tyr-αCD3-CD86-mut IL-2, pU57-Tyr-αCD3-CD86-mut IL-2-GFP, etc., using murine-derived DNA by a seamless cloning method.
The constructed plasmids were sequenced and the sequencing results were as follows:
outcome: The plasmids pU57-Tyr-mut IL-2 were successfully constructed
outcome: The plasmids pU57-Tyr-αCD3-CD86-mut IL-2 were successfully constructed
iCLAN-qPCR detection of gene expression:
(mut IL-2 relative mRNA expression) mut IL-2 sequence is highly transferred in B16 cells with transfection of iCLANTyr-mut IL-2 and iCLANTyr-αCD3-CD86+Tyr-mut IL-2.
(αCD3 relative mRNA expression) αCD3 is highly transferred in B16 cells with transfection of iCLANTyr-αCD3-CD86 and iCLANTyr-aCD3-CD86+Tyr-mut IL-2, while a small amount of αCD3 relative mRNA was observed with transfection of iCLAN Tyr-mut IL-2.
(CD86 relative mRNA expression) CD86 is highly transferred in B16 cells with transfection of iCLANTyr-αCD3-CD86 and iCLANTyr-aCD3-CD86+Tyr mut IL-2, while a small amount of CD86 relative mRNA was observed with transfection of iCLAN Tyr-mut IL-2.
During this engineering cycle, we identified a number of areas for improvement to further refine our plasmid:
(1) It is not enough to treat melanoma alone, we also searched for Survivin, a promoter that generally induces large amounts of transcription in a wide range of tumor cells.
(2) At the beginning of the experiment, we used IL-2, and after comparing the experiments, we found that mut-IL-2, which was mentioned in the newly researched literature, had better T cell proliferation and recruitment ability [1]. Therefore, we redesigned the plasmid from IL-2 fragment to mut-IL-2.
Common attributes of nucleic acid drugs, such as high molecular weight, negatively charged nature, and susceptibility to degradation by nucleases, exhibit limited bioavailability in the naked form, severely limiting their delivery. iCLAN (cationic lipid-assisted polymeric nanoparticles) are tiny vesicles composed of cationic lipids (positively charged lipids) that can be used in drug delivery systems, especially for a wide range of applications in gene therapy and mRNA therapy. Nanoparticles have been shown to be a promising drug delivery system for nucleic acids, and modulation of nanoparticle properties through passive, active, and endogenous targeting mechanisms can improve the organ selectivity of drug delivery. Cationic liposomes are able to bind negatively charged biomolecules, such as DNA and RNA, through charge interactions to form stable complexes that protect these macromolecules from enzymatic breakdown in the body and facilitate their uptake by cells.
We prepared iCLAN from PEG-PLGA, DOTAP and MC3, which are FDA-approved drug adjuvants with a certain degree of safety. PEG is not easily degraded, but its toxicity is low and biocompatible, and it can be excreted through the kidneys. DOTAP is a cationic lipid, which can be hydrolyzed by lipase and phosphatase to generate fatty acids and glycerol, and the fatty acids can be further metabolized into the β-oxidation pathway and eventually converted into acetyl coenzyme A to enter the citric acid cycle to generate energy. Fatty acids can be further metabolized into the β-oxidation pathway and eventually converted into acetyl coenzyme A, which enters the citric acid cycle to generate energy. Glycerol, on the other hand, can enter the gluconeogenic pathway.MC3 is an ionizable lipid that promotes nucleic acid encapsulation in the LNP and mediates the disruption of nuclear endosomal membranes, allowing nucleic acids to be released into the cytoplasm.
iCLAN particle size characterization
Outcome: the average diameter and potential of self-assembly nanoparticle is reasonable and concentrated.
iCLAN-WB verification of target gene expression
aCD3-CD86 proteins were successfully expressed on the cells, while mut IL-2, a kind of secreted protein, could only be detected inside the cell, which is unexpected.
IL-2 protein can be expressed with high efficiency.
mut IL-2 can only be observed inside B16 cells(cell lysis), which proves that, the plasmid has been transcribed and translated, but mut IL-2 is not secreted outside the cell. Next step: measure the concentration of mut IL-2 in supernate (ELISA).
(1) Through this test cycle we tried various different components and ratios of iCLAN, and finally determined the iCLAN synthesis pathway suitable for the genetic nanomedicine of this project.
(2) Various methods of plasmid encapsulation were tried, and a one-step method with a higher encapsulation rate was determined.
[1] Silva, D. A., Yu, S., Ulge, U. Y., Spangler, J. B., Jude, K. M., Labão-Almeida, C., Ali, L. R., Quijano-Rubio, A., Ruterbusch, M., Leung, I., Biary, T., Crowley, S. J., Marcos, E., Walkey, C. D., Weitzner, B. D., Pardo-Avila, F., Castellanos, J., Carter, L., Stewart, L., Riddell, S. R., … Baker, D. (2019). De novo design of potent and selective mimics of IL-2 and IL-15. Nature, 565(7738), 186–191. https://doi.org/10.1038/s41586-018-0830-7.